Device for detecting different fluorescence signals of a sample support illuminated with different excitation wavelengths

ABSTRACT

The invention relates to a device for detecting different fluorescence signals of a sample support illuminated with different excitation wavelengths. Said sample support comprises a number of samples that can be exited by different light wave-lengths for emitting differing fluorescent light. To this end, light sources ( 1, 3 ) are provided for generating rays of light or different excitation wavelengths, which can be directed toward the sample support ( 9 ) by excitation optics ( 5, 11 ). The fluorescent light ( 23, 45 ) emitted each time by the sample support ( 9 ) can be directed toward a receiver ( 25 ), which generates corresponding fluorescence signals. A mirror assembly ( 19 ) with reflective areas ( 19′ ) and transparent areas ( 19″ ) is connected between the different light sources ( 1, 3 ) and the sample support ( 9 ). Said mirror assembly can be displaced in such a manner that the ray of light of a light source ( 1 ) passes through a transparent area ( 19″ ) and reaches the sample support ( 9 ), whereas the rays of all other light wavelengths are reflected by reflective areas ( 19′ ) thereby preventing them from reaching the sample support ( 9 ).

[0001] Device for detecting different fluorescence signals of a samplecarrier illuminated by different excitation wavelengths

[0002] The invention relates to a device for detecting differentfluorescence signals of a sample carrier illuminated by differentexcitation wavelengths according to the preamble of patent claim 1.

[0003] Wherever quantitative fluorescence immunoassays, for example, arecarried out, sample carriers are known that have a multiplicity ofelectrodes, for example 10 000 electrodes, to which an electric voltagecan be applied selectively. If different sample liquids are led over theelectrodes, different samples can be produced by deposition at theelectrodes, depending on the application of specific voltages. Sincethese samples are marked by two or more fluorescence carriers, theyluminesce differently in the case of excitation by different opticalwavelengths. Biochemical properties can be measured in this way.

[0004] It is known in this connection to use dichroic, permanentlyinstalled mirrors in order to achieve a separation of the differentfluorescence wavelengths that are emitted by the sample carrier. In thiscase, a problem consists in that dichroic mirrors can be operated onlywhen the beam path is parallel to the position of the dichroic mirrors.In addition, such mirrors are not 100% efficient. At the same time, theyalso require the excitation sources to be electrically clocked.

[0005] Sample carriers produced using semiconductor technology are, forexample, built up in several layers and have a multiplicity ofcylindrical platinum electrodes to which it is possible to apply theabovenamed voltages. The sample carriers are arranged in plasticcontainers covered in each case with a glass layer, it being possiblefor the said sample liquids to flow through the space between the glasslayer and plastic container and come into contact in the process withelectrodes.

[0006] Document DE 39 26 090 C2 discloses a dual-beam photometer inwhich a rotatable mirror system divided into silvered and transmittingsectors is used to split a light bundle issuing from a light source intoa measuring beam and into a reference beam. The two beam paths arerecombined by the same mirror system, the measuring beam penetrating themirror system and passing through a sample to be examined, and thereference beam being reflected at the mirror system and therefore notimpinging on the sample. The recombined beam is detected by a detectordevice. Consequently, the influence of fluctuations in the light sourcebrightness or the detector sensitivity can be eliminated given suitableevaluation of the detected measuring signals. DE 39 26 090 C2 furtherdiscloses in accordance with an exemplary embodiment a dual-beamphotometer having a second light source emitting a continuous spectrum(see FIG. 4), whose radiation is used in a fashion alternating with thefirst light source both as measuring beam and as a reference beam when amirror system divided into four sectors (two silvered and twotransparent sectors) is used. It is possible in this way additionally toachieve compensation of background radiation.

[0007] The object of the present invention consists in creating a devicefor detecting different fluorescence signals of a sample carrierilluminated by different excitation waves, in the case of which it isnot necessary to clock the light sources generating the differentexcitation wavelengths.

[0008] This object is achieved by means of a device having the featuresof patent claim 1.

[0009] The advantage of the present invention consists in that in orderto detect different fluorescence signals of a sample carrier illuminatedby different, preferably two, excitation wavelengths, there is no needto use dichroic mirrors that have the disadvantages outlined above. Inthe case of the device according to the invention, the requiredseparation of the fluorescence signals is performed with the aid of arotatable mirror arrangement that is arranged in the beam path of theexcitation and detection optical system and is partly transmitting inaccordance with the number of the excitation wavelengths. This mirrorarrangement is moved in the beam path such that in each case oneexcitation detection channel is opened and the other excitationdetection channels are closed. It is particularly preferred for themirror arrangement to be a rotatable mirror.

[0010] Further advantageous refinements of the invention follow from thesubclaims. The invention and its refinements are explained in moredetail below in conjunction with the figures, in which:

[0011]FIG. 1 shows a schematic of a first side view of the detectionpart of the device according to the invention for detecting twodifferent fluorescence signals of a sample carrier illuminated by twodifferent excitation waves;

[0012]FIG. 2 shows a side view of the entire device of FIG. 1 at aviewing angle rotated by 90°, and

[0013]FIG. 3 shows a plan view of a rotatable mirror that is suitablefor a device for detecting two different fluorescence signals in thecase of two different excitation waves.

[0014] In accordance with FIGS. 1 to 3, a preferred device for detectingtwo different fluorescence signals in the case of two excitationwavelengths essentially has a first light source 1, a second lightsource 3, a first excitation optical system 5 that directs the firstlight beam 7 from the first light source 1 onto a sample carrier 9, asecond excitation optical system 11 that directs the second light beam13 from the second light source 3 onto the sample carrier 9, a firstfilter 15 assigned to a first fluorescent light, a second filter 17assigned to a second fluorescent light, as mirror arrangement asegmented rotatable mirror 19 that in accordance with FIG. 3 has a firsttransmitting region 19″ and a second reflecting region 19′, a firstfixed mirror 21 that reflects the first fluorescent light 23 to thereceiver 25, and a second fixed mirror 27 that leads the secondfluorescent light to the rotatable mirror 19 from which it is reflectedto the receiver 25.

[0015] The first light source 1 and the second light source 3 arepreferably laser sources, the first light source 1 generating, forexample, a laser light of wavelength 532 nm, and the second light source3 generating, for example, a laser light of wavelength 632 nm. Thefilters 15 and 17 are preferably steep-edge filters that either allowonly the first or the second fluorescent light to pass. The firstexcitation optical system 5 comprises a first stop 30 and a first lensarrangement 31 that generate from the first laser beam generated by thefirst light source 1 a first parallel beam 7, and a first deflectingmirror 33 that directs the parallel beam 7 onto the sample carrier 9 insuch a way that the latter is illuminated over its entire surface.

[0016] Correspondingly, the second excitation optical system 11comprises a second stop 34 and a second lens arrangement 35 thatgenerate a second parallel beam 13 from the second laser beam from thesecond light source 3, and a second fixed deflecting mirror 37 thatdirects the parallel beam 13 onto the sample carrier 9 in order toilluminate the entire surface of the latter.

[0017] The rotatable mirror 19 can be rotated about an axis 20 ofrotation and has the reflecting region 19′ and the transmitting region19″ that, in the case of the use of two light sources 1, 3 of twodifferent wavelengths, preferably correspond in each case to half thesurface of the circular rotatable mirror 19.

[0018] The detection optical system 42 comprises an optical imagingarrangement 43 that is arranged downstream of the sample carrier 9 andgenerates a parallel beam in each case from the first and secondfluorescent light 23 and 45, respectively, output by the sample carrier9, and an optical imaging arrangement 48 that is arranged upstream ofthe receiver 25 and projects the said parallel beams onto the entiresurface of the receiver 25. The filter 15 and the fixed mirror 27 aswell as the filter 17 and the fixed mirror 21 are part of the detectionoptical system 42.

[0019] The receiver 25 is preferably a CCD arrangement that, inaccordance with the number of samples of the sample carrier 9, hasphotosensitive elements that respectively generate a first or secondelectric fluorescence signal in accordance with their illumination bythe first or second fluorescent light 23 and 45, respectively. Thesefluorescence signals are led to an electronic evaluation system (notillustrated in more detail).

[0020] For example, the sample carrier 9 and the receiver 25 havesamples or optical sensor elements in mutually corresponding rasterconfigurations, the number of samples or sensor elements being of theorder of 10 000.

[0021] The function of the present device for separating twofluorescence signals is explained below in more detail.

[0022] It is assumed in this case that the reflecting region 19′ islocated in a phase in FIG. 1 to the left of the axis 20 of rotation, andthe transmitting region 19″ is located to the right of the axis 20 ofrotation. The result of this is that the first laser beam 7 generated bythe first light source 1 passes through the transmitting region 19″ andis led to the sample carrier 9 by the imaging optical system 5 (FIG. 2)in order to illuminate the entire surface of the latter. The firstfluorescent light 23 emitted by the sample carrier 9 as a consequence ofthe wavelength of the first laser beam 7 is reflected at the reflectingregion 19′ and directed onto the filter 17, passes through the latter,is reflected at the fixed mirror 21, passes through the transmittingregion 19″ of the mirror 19 (FIG. 1) and is directed by the imagingoptical system 48 onto the receiver 25, which generates correspondingfluorescence signals at its individual photosensitive sensor elements.During this phase, the laser beam 13 emitted by the second light source3 is reflected at the reflecting region 19′ such that it cannot reachthe second deflecting mirror 37 and cannot reach the sample carrier 9(FIG. 2).

[0023] In the other phase, in which the reflecting region 19′ is locatedto the right of the axis 20 of rotation, and the transmitting region 19″is located to the left of the axis 20 of rotation, the second laser beam13 from the light source 3 passes through the transmitting region 19″and is directed by the second deflecting mirror 37 onto the samplecarrier 9 (FIG. 2 with interchanged regions 19′, 19″). The secondfluorescent light 45 generated in this case passes through thetransmitting region 19″, passes the filter 15, is reflected by the fixedmirror 27 to the reflecting region 19′ of the rotatable mirror 19 and isreflected at the latter and directed to the imaging optical system 44(FIG. 1, dotted lines). The latter projects the second fluorescent light45 onto the receiver 25. The individual optical sensor elements of thereceiver 25 then generate corresponding second fluorescence signals. Inthis phase, the first laser beam generated by the first light source 1is reflected at the reflecting region 19′ such that it cannot reach thefirst deflecting mirror 33 and also cannot reach the sample carrier 9.

[0024] It is possible in this way to use the rotary movement of therotatable mirror 19 to switch back and forth between the two laser beams7 and 13, which are generated simultaneously, in order respectively tobe able to illuminate the entire surface of the sample carrier 9, suchthat in each case only one laser beam illuminates the sample carrier 9and a fluorescent light is generated that is led to the receiver 25,while the respective other laser beam is reflected at the reflectingregion 19″ of the rotatable mirror 19 such that it cannot reach thereceiver 25. Consequently, the different fluorescence signals arereceived in successive sequence at the receiver 25 and, if the receiver25 is a CCD arrangement, are latched to an electronic evaluation device.

[0025] It may be pointed out that in order to separate more than twofluorescence signals it is also possible for the rotatable mirror 19 tohave a plurality of transparent and reflecting regions so as to ensurethat in different phases it is always only one fluorescent light that isexcited by a laser beam and led to the receiver, while the respectiveother laser beams are reflected at the reflecting regions such that theycannot excite fluorescent light.

[0026] It is also possible to use other movable mirror arrangementinstead of the rotatable mirror 19 explained. For example, a transparentand reflecting regions can be moved back and forth next to one anotherin a plate having row, this being done in the direction of the row.

1. A device for detecting different fluorescence signals of a sample carrier illuminated over its entire surface by different excitation wavelengths, which has a plurality of samples that can be excited by different optical wavelengths to output different fluorescent light, in which a first light source (1) and a second light source (3) are provided which generate light beams (7, 13) of different optical wavelengths, which are directed via an excitation optical system (5, 11) in each case to the entire surface of the sample carrier (9), in the case of which the fluorescent light (23, 45) respectively emitted by the sample carrier (9) is directed to the receiver (25) generating corresponding fluorescence signals in such a way that each sample is projected onto a photosensitive element, corresponding to the respective sample, of the receiver (25), and in the case of which a mirror arrangement (19) with silvered regions (19′) and transmitting regions (19″) is connected between the different light sources (1, 3) and the sample carrier (9) and is capable of moving such that the first light beam (7) of the first light source (1) passes through the transmitting region (19″) to the sample carrier (9), and the second light beam (13) from the second light source (3) is reflected from the reflecting region (19′) such that it does not reach the sample carrier (19), and the first fluorescent light (23) generated by the first light beam (7) is emitted to the reflecting region (19″), which reflects it to a first fixed mirror (21), and that the first fixed mirror (21) reflects the first fluorescent light (23) through the transmitting region (19″) to the receiver (25), the receiver (25) being screened from the light sources (1, 3) in such a way that the receiver (25) exclusively detects fluorescent light emitted by the samples.
 2. The device as claimed in claim 1, characterized in that the mirror arrangement (19) is a mirror that can be rotated about an axis (20).
 3. The device as claimed in one of claims 1 to 2, characterized in that the first light beam (7) can be directed by a first excitation optical system (5) to the sample carrier (9) in order to illuminate the entire surface of the latter.
 4. The device as claimed in one of claims 1 to 3, characterized in that the second light beam (13) can be directed by a second excitation optical system (11) to the sample carrier (9) in order to illuminate the entire surface of the latter.
 5. The device as claimed in one of claims 1 to 4, characterized in that the second fluorescent light (45) generated by the second light beam (13) passes from the sample carrier (9) through the transmitting region (19″) of the mirror (19), and is reflected from a second fixed mirror (27) onto the reflecting region (19′) of the mirror (19) and is reflected from the reflecting region (19′) of the mirror (19) to the receiver (25).
 6. The device as claimed in one of claims 2 to 5, characterized in that the rotatable mirror is of circular design and has a semicircular transmitting region (19′) and a semicircular reflecting region (19′).
 7. The device as claimed in one of claims 3 to 6, characterized in that the first excitation optical system (5) has a first deflecting mirror (33) that directs the first light beam (7) to the sample carrier (9).
 8. The device as claimed in one of claims 4 to 7, characterized in that the second excitation optical system (11) has a second deflecting mirror (37) that directs the second light beam (13) to the sample carrier (9).
 9. The device as claimed in one of claims 1 to 8, characterized in that provided between the sample carrier (9) and the receiver (25) in the beam path of the first fluorescent light (23) is a first filter (17), which passes only the first fluorescent light (23).
 10. The device as claimed in one of claims 5 to 9, characterized in that there is provided in the beam path of the second fluorescent light (45) between the sample carrier (9) and the receiver (25) a second filter (15), which passes only the second fluorescent light.
 11. The device as claimed in claim 9 or 10, characterized in that the first filter (17) and/or the second filter (15) is a steep-edge filter.
 12. The device as claimed in one of claims 1 to 11, characterized in that the receiver (25) is a CCD arrangement that has for the purpose of generating electric fluorescence signals from the received first or second fluorescent light (23, 45) optical sensor elements that are arranged in accordance with a specific arrangement of the samples of the sample carrier (9), the electric fluorescence signals generated at the sensor elements during illumination by a fluorescent light being latched to an electronic evaluation system before the illumination by the other fluorescent light. 